Allosteric Stabilization of3H-N-Methylscopolamine Binding in Guinea-Pig Myocardium by an Antidote against Organophosphate Intoxication

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Abstract
W84 (hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide]) protects overadditively against an organophosphate-intoxication when applied in combination with atropine. Further experimental evidence led to the hypothesis that W84 exerted an allosteric effect on muscarinic acetylcholine receptors. In order to investigate the action of W84 on the receptor level, binding studies with 3H-N-methylscopolamine were performed in homogenized and intact guinea-pig myocardium. For sake of comparison three bispyridinium oximes were included, i.e. Uno3 (trimethylene-bis-[4-hydroxyiminomethyl-pyridinium] dibromide mono-2,6-dichlorobenzylether), obidoxime, and TMB4. In cardiac membrane suspensions, all compounds inhibited 3H-NMS-binding after 2 hrs of incubation concentration-dependently by reducing its affinity, whereas leaving the number of binding sites unaltered. However, with increasing concentrations W84 suppressed 3H-NMS-binding less than expected for a competitive antagonist. Kinetic studies revealed that W84 did not only slow the association of 3H-NMS, but additionally retarded its dissociation over the entire range of concentrations that inhibited 3H-NMS-binding. At 1 mM, W84 augmented the half time of the 3H-NMS-receptor complexes from a control values of 4 min to more than 120 min. The stabilization of the radioligand-receptor complexes is indicative of an allostric effect of W84. Obidoxime, TMB4 and Uno3 at low concentration acted like competitive inhibitors of 3H-NMS-binding. From 10-5M onwards, Uno3 retarded 3H-NMS-dissociation concentration-dependently. It is concluded that the effect of bisquaternary compounds on 3H-NMS-association and -dissociation is mediated via binding to two separate sites, i.e. the muscarinic receptor site and an allosteric effector site, respectively. Also in intact beating left atria, W84 retarded 3H-NMS-dissociation concentration-dependently. The stabilizing effect of W84 on antagonist-receptor complexes may explain the overadditive protective action exerted by the combination of atropine and W84 against organophosphate intoxication.